Endpoint TaqMan methods for determining zygosity of cotton comprising Cry1Ac event 3006-210-23

Abstract

A method for zygosity analysis of the cotton Cry1Ac event 3006-210-23 is provided. The method provides 3006-210-23 event-specific and cotton-genome-specific primers and TaqMan probe combinations for use in an endpoint biplex TaqMan PCR assay capable of determining event zygosity and for assisting in event introgression and breeding.

The invention claimed is:  
     
       1. A method for determining event zygosity of a cotton plant comprising a 3006-210-23 event, said 3006-210-23 event comprising a transgene construct comprising a cry1Ac gene, said method comprising:
 obtaining a sample of genomic DNA from said cotton plant, 
 contacting said sample with
 a. a first flanking primer consisting of SEQ ID NO:2; 
 b. a second flanking primer consisting of SEQ ID NO:4; 
 c. a transgene primer consisting of SEQ ID NO:3; 
 
 wherein said first flanking primer and said second flanking primer form a wild-type amplicon when subjected to PCR conditions, 
 wherein said transgene primer forms a transgene amplicon with said first flanking primer or said second flanking primer when subjected to PCR conditions, 
 further contacting said sample with
 d. a florescent event probe that hybridizes with said transgene amplicon 
 e. a florescent wild-type probe that hybridizes with said wild-type amplicon 
 
 subjecting said sample to fluorescence-based endpoint TaqMan PCR conditions, quantitating said florescent event probe that hybridized to said event amplicon, 
 quantitating said florescent wild-type probe that hybridized to said wild-type amplicon, 
 comparing amounts of hybridized florescent event probe to hybridized florescent wild-type probe; and 
 determining zygosity of said cotton tissue by comparing florescence ratios of hybridized fluorescent event probe and hybridized fluorescent wild-type probe. 
 
     
     
       2. The method of  claim 1 , wherein said plant comprises a first subgenome and a second subgenome, said transgene amplicon being formed from said first subgenome, and said wild-type amplicon being formed from said second subgenome. 
     
     
       3. The method of  claim 1  wherein results of said method are read directly in a plate reader. 
     
     
       4. The method of  claim 1  wherein said probes are labeled with a fluorescent dye and quencher. 
     
     
       5. The method of  claim 1  wherein said transgene probe comprises TEXAS RED™ as said fluorescent dye at the 5′ end of said transgene probe and BLACK HOLE QUENCHER (BHQ™) as said quencher on the 3′ end of said transgene probe. 
     
     
       6. The method of  claim 1  wherein said wild-type probe is labeled with a fluorescent dye (FAM) at the 5′ end of said wild-type probe and a BLACK HOLE QUENCHER (BHQ™) as said at the 3′ end of said wild-type probe. 
     
     
       7. The method of  claim 1  wherein said wild-type probe comprises SEQ ID NO:6. 
     
     
       8. The method of  claim 1  wherein said transgene probe comprises SEQ ID NO:5. 
     
     
       9. The method of  claim 1  wherein said transgene amplicon comprises SEQ ID NO:8. 
     
     
       10. The method of  claim 1  wherein said wild-type amplicon comprises SEQ ID NO:9.